Eliane Isabelle Marti

Analysis of diversity and population structure in the Lipizzan horse breed based on pedigree information

The pedigrees of 565 Lipizzan horses from eight European studs were traced back to the individuals considered as the founding population of the breed. The length of pedigrees was up to 32 generations, with an average of 15.2 complete generations. Average inbreeding coefficients varied from 8.6 to 14.4% between studs, with an overall mean of 10.8%. Increase in inbreeding was relatively small in the last generation. Other measures of genetic variability based on contributions of founders or important ancestors were also calculated, leading to partly different conclusions about the variability of the population at stud level. Average coancestries between studs showed that the two Romanian studs were most similar but also that the Austrian, Slovenian and Italian studs formed a cluster of genetically similar individuals. The pedigrees were also used to calculate gene proportions of founder breeds and individual founders. About 52% of the current genes are of Spanish or Italian origin, 21% Arabian, 8% Fredriksborg, 4% Kladruby, 3% English and 2% Shagya-Arab. The most important founder individual was Toscanello Hedera, followed by Neapolitano, one of the founders of the classical stallion lines, each contributing more than 6% of the current gene pool.

Allergen-specific IgE levels against crude mould and storage mite extracts and recombinant mould allergens in sera from horses affected with chronic bronchitis.

Immunoglobulin E antibody (IgE) levels against four recombinant (r) mould allergens (r-Aspergillus fumigatus [rAsp f] 7, 8 and 9; r-Alternaria alternata 1 [rAlta1]) and crude mould (Aspergillus fumigatus, Alternaria alternata, Penicillium notatum) and storage mite extracts were determined by ELISA in sera from 24 pulmonary sound control horses and 26 horses suffering from chronic bronchitis/bronchiolitis (CB), also called chronic obstructive pulmonary disease (COPD). Serum IgG and IgA titres were also determined against Aspergillus fumigatus extract and rAsp f 8.IgE against the crude extracts could be measured in all sera, but there was no significant difference between CB-affected and control horses. In contrast, only 8-30% of the horses, depending on the r-allergen tested, had detectable IgE levels in serum against the r-allergens. Horses with CB had significantly more often detectable IgE levels than controls against rAlt a 1 (10/26 and 3/24, respectively, p=0. 054), rAsp f 7 (13/26 and 2/24, respectively, p<0.01) and rAsp f 8 (11/26 and 1/24, respectively, p<0.01). Only four horses (three CB-affected and one healthy, p0.05) had detectable IgE levels against rAsp f 9. Furthermore, CB-affected horses were often sensitised against two or more r-allergens (13/26 of the CB-affected horses) while only one of the 24 healthy horses had positive IgE levels against more than one r-allergens. Similarly to IgE levels, no significant differences between CB-affected and healthy horses were found for IgG titres against the Aspergillus fumigatus extract. However, horses with CB had significantly higher serum IgG titres against rAsp f 8 than healthy controls (median=28 versus 10 relative ELISA units [REU], p<0.01). Additionally, horses with detectable IgE titres against rAsp f 8 had significantly higher IgG titres against this r-allergen than horses with undetectable IgE titres (median IgG titres=46 and 13 REU, respectively; p<0.01). For serum IgA titres, neither differences between healthy and CB-affected animals nor correlations between IgA and IgG or IgE titres could be found. These results show that horses suffering from CB are more often sensitised to some Aspergillus fumigatus and Alternaria alternata allergens than control horses and that they are partly sensitised to the same fungal proteins as mould-allergic human patients. Furthermore, this study shows that r-allergens allow a much more sensitive determination of specific serum antibody levels by ELISA than crude mould extracts.

Detection of IgG and IgE serum antibodies to Culicoides salivary gland antigens in horses with insect dermal hypersensitivity (sweet itch)

We postulated that all horses exposed to the bites of Culcoides (midges) would have an antibody response to the antigen secreted in Culcoides saliva, but that IgE antibody would be restricted to allergic individuals. Using immunohistology on sections of fixed Culicoides, we have demonstrated the presence of antibodies in horse serum which recognise Culicoides salivary glands. Antibodies were detected in the serum of horses with insect dermal hypersensitivity and in the serum of normal horses exposed to Culicoides bites. In contrast, no antibodies were detected in serum from native Icelandic ponies which had not been exposed to Culicoides. Anti-salivary gland IgG antibodies were detected in serum from both allergic and healthy horses exposed to Culicoides. IgE antibodies were only detected in horses with signs of insect dermal hypersensitivity, they were not found in serum of healthy controls nor in the serum of horses with a history of hypersensitivity but in remission at the time of sampling. Using western blotting we confirmed the presence of antibodies to Culicoides antigens and demonstrated that individual horses react to different numbers of antigens. This paper demonstrates the ability of serum from allergic horses to detect Culcoides antigens and will enable further studies to isolate and characterise the allergens.

Airway inflammation and mucus in two age groups of asymptomatic well-performing sport horses.

REASONS FOR PERFORMING STUDY Horses housed in conventional stable environments are exposed to high dust loads. Long-term exposure to inhaled dust may lead to subclinical airway disease. HYPOTHESES Airway inflammation and mucus accumulation are 1) common in asymptomatic well-performing sport horses, 2) increased in older compared to younger individuals and 3) correlated. METHODS Mucus quantity and quality (endoscopic scoring) and BALF differential cytology were assessed in 13 younger horses (mean age 5 years) and 13 older horses (mean age 15 years), which had no historical or clinical evidence of lower airway disease, were performing well and were housed permanently in a conventional stable environment. RESULTS 1) Airway inflammation and/or mucus accumulation were very common. All sport horses showed evidence of subclinical inflammatory airway disease (IAD). 2) Older horses, having been exposed for 10 more years to a conventional stable environment, did not show increased subclinical airway inflammation or mucus accumulation. The only differences between the age groups were increased BALF total and lymphocyte cell counts in the younger horses. 3) Mucus quantity and quality scores were not significantly correlated with each other or with BALF neutrophil percentages and absolute numbers. CONCLUSIONS Although clinically healthy and performing well, all of the examined horses housed in a conventional stable environment showed evidence of IAD. The degree of IAD was not increased in older horses that had not developed clinical signs. POTENTIAL RELEVANCE The effect of subclinical degrees of IAD on performance in dressage and show jumping should be investigated further to better understand the clinical significance of IAD in sport horses.

Effects of genetic and environmental factors on chronic lower airway disease in horses

BACKGROUND Environment and genetics influence the manifestation of recurrent airway obstruction (RAO), but the associations of specific factors with mild, moderate, and severe clinical signs are unknown. HYPOTHESIS We hypothesized that sire, feed, bedding, time outdoors, sex, and age are associated with clinical manifestations of mild, moderate, and severe lower airway disease. ANIMALS Direct offspring of 2 RAO-affected Warmblood stallions (F1S1, n = 172; F1S2, n = 135); maternal half-siblings of F1S1 (mHSS1, n = 66); and an age-matched, randomly chosen control group (CG, n = 33). METHODS A standardized questionnaire was used to assess potential risk factors and to establish a horse owner assessed respiratory signs index (HOARSI 1-4, from healthy to severe) according to clinical signs of lower airway disease. RESULTS More F1S1 and F1S2 horses showed moderate to severe clinical signs (HOARSI 3 and HOARSI 4 combined, 29.6 and 27.3%, respectively) compared with CG and mHSS1 horses (9.1 and 6.2%, respectively; contingency table overall test, P < .001). Sire, hay feeding, and age (in decreasing order of strength) were associated with more severe clinical signs (higher HOARSI), more frequent coughing, and nasal discharge. CONCLUSIONS AND CLINICAL RELEVANCE There is a genetic predisposition and lesser but also marked effects of hay feeding and age on the manifestation of moderate to severe clinical signs, most markedly on coughing frequency. In contrast, mild clinical signs were not associated with sire or hay feeding in our populations.

Equine insect bite hypersensitivity: what do we know?

Insect bite hypersensitivity (IBH) is an allergic dermatitis of the horse caused by bites of insects of the genus Culicoides and is currently the best characterized allergic disease of horses. This article reviews knowledge of the immunopathogenesis of IBH, with a particular focus on the causative allergens. Whereas so far hardly any research has been done on the role of antigen presenting cells in the pathogenesis of IBH, recent studies suggest that IBH is characterized by an imbalance between a T helper 2 (Th2) and regulatory T cell (T(reg)) immune response, as shown both locally in the skin and with stimulated peripheral blood mononuclear cells. Various studies have shown IBH to be associated with IgE-mediated reactions against salivary antigens from Culicoides spp. However, until recently, the causative allergens had not been characterized at the molecular level. A major advance has now been made, as 11 Culicoides salivary gland proteins have been identified as relevant allergens for IBH. Currently, there is no satisfactory treatment of IBH. Characterization of the main allergens for IBH and understanding what mechanisms induce a healthy or allergic immune response towards these allergens may help to develop new treatment strategies, such as immunotherapy.

Morphological description of the Lipizzan horse population

Abstract Morphological measurements of Lipizzan horses from state-owned studs in seven countries in Central and South-east Europe (Austria, Croatia, Hungary, Italy, Romania, Slovakia and Slovenia) were recorded to characterise the base population of Lipizzan horse breeding. A total of 37 distance and angular measures were taken from 368 breeding mares and 145 stallions from eight studs (one stud from each country with the exception of Romania, where horses were measured at two studs). Significant differences between studs were found for most of the traits (34 of 37 for mares, 29 of 37 for stallions). Repeatabilities of measurements (based on two repetitions in 100 horses) varied from 0.24 to 0.95. Some of the measurements most frequently taken in horse breeding showed high repeatabilities (0.95 for height at withers and cannon bone circumference), but chest girth showed a rather low repeatability of 0.45. Heritabilites of the traits varied from 0.00 to 0.55. Estimates were comparable to literature results but due to the small data set (only the 368 mares were used for analysis) they were partly inconsistent. Multivariate analyses were applied separately to mares and stallions. Length of neck, cannon bone circumference, and width of thorax were the most important traits for discriminating studs for mares; width of thurls, chest circumference, and width of hips were important for stallions. Mahalanobis distances based on all 37 measurements were significant for pairwise comparison of all studs. An analysis of the differences of sire lines showed no significance. The results reflect at least partially different breeding goals.

Evaluation of an in vitro sulphidoleukotriene release test for diagnosis of insect bite hypersensitivity in horses

REASONS FOR PERFORMING STUDY Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis caused by bites of Culicoides and Simulium species, and improved means of diagnosis are required. OBJECTIVES The cellular antigen simulation test (CAST) with C. nubeculosus and S. vittatum extracts was assessed in a population of IBH-affected and healthy horses. Variations in test results over a one year period and possible cross-reactivity between different insect extracts was studied. METHODS A total of 314 mature horses were studied using the CAST. Influence of severity of clinical signs, gender and age were evaluated, and 32 horses were tested repeatedly over one year. The kappa reliability test was used to assess agreement of the test results with different insect extracts. RESULTS Horses with IBH had significantly higher sLT release than controls with C. nubeculosus and S. vittatum. The highest diagnostic sensitivity and specificity levels were attained when using adult C. nubeculosus extracts with the CAST (78% and 97%, respectively), suggesting that most horses with IBH are sensitised against Culicoides allergens. A proportion of IBH-affected horses was found to be sensitised to allergens of Simulium spp. in addition to those of C. nubeculosus. The CAST with C. nubeculosus had positive and negative predictive values > or = 80% for a true prevalence of IBH of 12-52%. In the follow-up study, the proportion of IBH-affected horses with a positive test result ranged from 90% in November to 68% in March. Severity of clinical signs or age did not influence test results significantly. However, IBH-affected males achieved significantly more positive test results than IBH-affected females. CONCLUSIONS The CAST with adult C. nubeculosus has high specificity and good sensitivity for diagnosis of IBH. Horses with IBH are mainly sensitised to Culicoides allergens, and some horses are additionally also sensitised to allergens in Simulium spp. POTENTIAL RELEVANCE The CAST is likely to be a useful test for diagnosis of IBH, even allowing the identification of IBH-affected but asymptomatic horses. This test may also help in further characterisation of allergens involved in this condition.

Influence of environmental and genetic factors on allergen-specific immunoglobulin-E levels in sera from Lipizzan horses

To investigate whether allergen-specific IgE production is influenced by environmental and genetic factors, IgE levels against 2 mould extracts (Alternaria alternata [Alt a] and Aspergillus fumigatus [Asp f]) and against recombinant (r) rAlt a 1, rAsp f 7 and rAsp f 8 were determined by ELISA in sera from 448 Lipizzan horses living in 6 studfarms. Statistical evaluation showed a significant effect of studfarm-specific environment on IgE levels against the different allergens, but genetic factors also influenced allergen-specific IgE production: an heritability of 0.33 was found for IgE levels against the 2 mould extracts and of 0.21 for rAsp f 8-specific IgE. Heritability estimates for rAlt a 1- and rAsp f 7-specific IgE were negligible. Investigations for a possible association between Major Histocompatibility Complex (MHC) class I antigens and specific IgE levels were carried out. The most consistent significant association was found between the equine leucocyte antigen (ELA) A8 and undetectable IgE titres against rAsp f 7 and rAsp f 8. Significant ELA associations were also demonstrated between ELA A1 and higher specific IgE levels, between ELA A14 and lower IgE levels against the mould extracts and in one studfarm between ELA Be27 and lower Aspergillus-specific IgE levels.

Immunoglobulin-E-bearing cells in skin biopsies of horses with insect bite hypersensitivity

The aim of the present study was to investigate, with immunohistochemistry and in situ hybridisation, if immunoglobulin-E (IgE) and mast cells are involved in the pathogenesis of insect bite hypersensitivity (IBH), an allergic dermatitis of horses. In tissue sections fixed in paraformaldehyde (PFA) for <24 h, significantly more IgE protein-bearing cells were found in the dermis and epidermis of acute and chronic IBH lesions than in skin biopsies from healthy horses (medians = 466, 236 and 110 cells/mm2, respectively; P < or = 0.01). More IgE-mRNA positive (+) cells were observed in the dermis of acute IBH lesions than in the dermis of healthy skin (median = 2.8 vs. 0.0 cells/mm2; P < or = 0.01). Significantly, more mast cells were detected with metachromatic (median = 160 vs. 62 cells/mm2; P < or = 0.001) and tryptase-specific stainings (median = 120 vs. 69 cells/mm2; P < or = 0.001) in the dermis of acute IBH biopsies compared to healthy skin. No chymase+ mast cells were found in any skin biopsy. IBH lesions fixed in PFA for >24 h were compared to dermatomycosis (DM) lesions; IBH biopsies contained a similar number of IgE-protein+ cells to DM biopsies (median = 249 vs. 192 cells/mm2; P = 0.08) but had significantly more IgE-mRNA+, metachromatic and tryptase+ mast cells than DM biopsies. This study suggests an involvement of IgE-mediated immune reactions in the pathogenesis of IBH as well as, sometimes, in dermatomycosis. Using double labelling, cells which expressed IgE protein and contained mast cell enzymes were detected.